국립 암 연구소 부여 1R03CA172923에 의해 부분적으로 지원됩니다.

윤리 정책 : 연구 마우스의 사용은 기본적인 생물학 또는 질병의 최종 처리에 대한 인간의 이해를 발전 있습니다 경우에만 허용됩니다. 1. 주문 <ol><li> 구매 여성 C57BL / 6 마​​우스 (6 세 - 8 주), 그룹 당 5 마우스. 쥐에 대한 몇 가지 일 조정할 수 있습니다. </li></ol> B16-BL6 셀의 제조 2 <ol><li> 멸균 후드에서 37도 인큐베이터과 장소에서 세포 배양 플레이트를 제거합니다. 플레이트는 B16-BL6 생쥐 흑색 종 세포와 약 70 %의 합류해야합니다. 더 큰 합류는 세포를 전이 가능성을 변경할 수 있습니다. </li><li> 1 분 동안 앉아 보자, 접시 당 1 ml의 0.05 %의 트립신 - EDTA를 추가 한 다음 트립신 미디어 솔루션을 대기음. </li><li> 접시 당 트립신 1 ML을 추가하고 10 분 동안 인큐베이터에 번호판을 반환합니다. </li><li> 멸균 후드로 인큐베이터에서 접시를 이동 한 후, 무 혈청 Rosewell 공원 기념 연구소 Mediu 4를 가하여각각의 판에 m (RPMI). </li><li> 멸균, 전동 피펫 세포를 수집합니다. 세포를 파괴하는, 아주 약간의 각도를 누르면 끝으로, 플레이트의 바닥에 전지를 꺼냅니다. 그렇지 토출 바늘 막힘 수도 세포 덩어리의 적절한 분리를 보장하기 위해 여러 번 반복. 회상과 50 ML 원뿔 튜브에 전송할 수 있습니다. </li><li> 셀 밀도를 결정하는 혈구를 사용합니다. 상수 B16-BL6 셀의 총 개수를 유지하는 무 혈청 RPMI의 양을 결정하는 것은 0, 0.065, 0.015, 0.25 및 0,500,000 세포 / ㎖의 최종 농도에 도달 할 필요가 있었다. </li><li> 마찬가지로 15 ML 원뿔 튜브에 50 ML 튜브에 미디어 나누어지는. 5 분 동안 2,250 XG의 원추형 원심 분리 튜브. 미디어를 가만히 따르다. 무 혈청 RPMI의 적절한 볼륨을 추가하고 hemacytometer를 통해 원하는 세포 밀도를 확인합니다. 추가 RPMI을 추가하거나 회전하고 그에 따라 작은 볼륨 -에 재현 탁하여 densities- 조정합니다. </li><li> 나누어지는 500 μL얼음에 표시된 1.8 ml의 튜브에 각각의 세포 현탁액. </li></ol> 3. 꼬리 정맥 주입 <ol><li> 꼬리에 의해 개최 마우스를 가지고, 마우스 제한 수단으로 후방 먼저 안내합니다. 그것은 외부의 꼬리와 제한 수단의 주요 챔버의 몸통으로, 제한 수단의 끝으로 마우스를 잡아 당깁니다. 마우스가 고정 될 때까지 밀어 계속의 제한 수단 플런저를 삽입합니다. </li><li> 측면 꼬리 정맥 중 하나가 위쪽으로 향하도록 90도 등 마우스를 회전합니다. 알코올 패드를 사용하여 적극적으로 꼬리 정맥을 가장 잘 보이는 곳에 특히 마우스의 꼬리 쪽 청소. 참고 : 좋은 조명이 B57BL / 6J 마우스에 효과적인 꼬리 정맥을 시각화 필수적이다. 가열 램프 나 가열 수술 패드는 불필요하지만, 또한 꼬리 정맥의 체적을 증가시킴으로써 도울 것이다. </li><li> 멸균 배양 후드에서 2 백만 세포 / ㎖ 튜브에서 세포 배양 배지 300 μl를 수집한다. 의 S를 쓸어 넘겨, 바늘 전환IDE와 주사기에서 거품을 배출, 플런저를 밀어. 250 μL의 최종 부피가 발생하는 문화 RPMI를 꺼냅니다. </li><li> 비 지배적 인 손으로 마우스 꼬리를 확장합니다. 비 - 지배적 인 손의 집게 손가락으로 상승 꼬리의 기단부와 엄지 손가락으로 눌려 꼬리의 말단부와 꼬리를 잡고. 주 : 이러한 방식으로, 꼬리 정맥을 횡 방향 위치에 유지하고, 꼬리의 더 선단 부분에서 가능하면 일치하는 엔트리와 상기 제한 수단의 표면에 평행. </li><li> 우선 분 하향 각 말단부를 향해 꼬리 정맥에 주사 바늘을 삽입하고 (바늘을 주사기의 상대 플런저 단부 저하) 상기 삽입시의 꼬리 정맥의 각도와 일치하도록 바늘을 조정 . </li><li> 꼬리 정맥에 바늘을 약 1cm를 삽입합니다. 삽입 후, 바늘이 정상 들고 플런저를 푸시하기 시작한다. 주 : 바늘 꼬리 정맥에있는 경우 및 바늘 끝이 N이고오티는 미디어가 정맥에 방해받지 않고 흘러해야 방해. 효과적인 주사를 정맥에서 혈액 통관을 특징으로한다. 이 저항 또는 기포 주입의 부위에서 형성되기 시작하면, 바늘을 제거하고, 꼬리 정맥을 따라 더욱 인접 위치에서 다시 시도. </li><li> 정맥에서 바늘을 제거하고 폐기합니다. 어떤 출혈을 멈추게하는 항목 사이트에 거즈를 잡습니다. </li><li> 제한 수단에서 플런저를 제거하고받는 사람 케이지에 마우스를 놓습니다. </li><li> 다른 모든 농도에 대해 반복합니다. 참고 :. 폐 초점 설립은 약 2 소요 - 변화 셀 라인과 초점의 원하는 크기에 따라 함께 3 주 (9)를 중간에서 마우스 과도한 헥헥처럼, 초기 안락사를 보증 할 수 증상을 모니터링해야한다. </li></ol> 4. 폐 초점을 계산 <ol><li> CO 2를 공동 2 챔버에 배치하고 100 % 노출하여 마우스를 안락사 </sUB는&gt; 5 분 동안 분 당 챔버 체적이 10 ~ 30 %로 도입했다. 자궁 전위에 따릅니다. </li><li> 스티로폼에 마우스를 스플레이. 수술 가위를 사용하여 흉골을 따라 수술 절개를 만들 수 있습니다. </li><li> 흉골 절개의 상단에서와 흉곽을 노출, 마우스의 어깨쪽으로 분기 모두 두 개의 추가 절개를합니다. </li><li> 폐와 심장을 노출, 몸통에서 갈비뼈를 제거하기 위해, 두 컷, 흉골의 측면을 따라 각합니다. </li><li> 수술 핀셋으로 마음을 잡고, 마우스에서 심장과 폐를 확보, 식도와 기관지를 잘라. 더 나은 시각화를 위해, 10 배 배율에서, 해부 현미경 아래에 배치합니다. </li><li> 단지 두 개의 폐를 떠나, 폐에서 심장을 해부 및 흉선을 제거합니다. </li><li> 해부 범위 아래에있는 폐와 폐의 초점을 계산하기 시작합니다. 각각의 초점은 폐의 표면에 원형, 갈색 강요로 나타납니다. <ol><li> 에 대한생쥐 간 일관성은 로브 (lobe)의 표면에 병소의 수를 카운트하고, 그 로브 반전. 네 개의 로브의 각 이렇게하면 개별적으로 쉽게 계산이 가능합니다. </li></ol></li><li> 유골을 폐기, 그렇지 않으면, 저장하고 IHC를 수행하는 경우 폐를 수정합니다. </li></ol>

<ol>
	<li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Kalluri, R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> J Clin Invest. 119 (6), 1420-1428 (2009).</li><li>Stetler-Stevenson, W. G., Aznavoorian, S., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+interactions+with+the+extracellular+matrix+during+invasion+and+metastasis.">Tumor cell interactions with the extracellular matrix during invasion and metastasis.</a> Annu Rev Cell Biol. 9, 541-573 (1993).</li><li>Allard, W. J., Matera, J., Miller, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cells+circulate+in+the+peripheral+blood+of+all+major+carcinomas+but+not+in+healthy+subjects+or+patients+with+nonmalignant+disease.">Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant disease.</a> Clin Cancer Res. 10, 6897-6904 (2004).</li><li>Dunn, G. P., Bruce, A. T., Ikeda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunoediting:+from+iimmunosurveillance+to+tumor+escape.">Cancer immunoediting: from iimmunosurveillance to tumor escape.</a> Nat Immunol. 3, 991-998 (2002).</li><li>Hugo, H., Ackland, M. L., Blick, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+and+mesenchymal-epithelial+transition+in+carcinoma+progression.">Epithelial-mesenchymal and mesenchymal-epithelial transition in carcinoma progression.</a> J Cell Physiol. 213, 374-383 (2007).</li><li>Kang, Y., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions:+Twist+in+development+and+metastasis.">Epithelial-mesenchymal transitions: Twist in development and metastasis.</a> Cell. 118, 277-279 (2004).</li><li>Hodi, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+survival+with+Ipilimumab+in+patients+with+metastatic+melanoma.">Improved survival with Ipilimumab in patients with metastatic melanoma.</a> NEJM. 363 (13), 711-723 (2002).</li><li>Ekwueme, D. U., Guy, G. P., Li, C., Rim, S. H., Parelkar, P., Chen, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+health+burden+and+economic+costs+of+cutaneous+melanoma+motality+by+race/ethnicity-United+States.">The health burden and economic costs of cutaneous melanoma motality by race/ethnicity-United States.</a> J. Am. Acad. Dermatol. 65, 133-143 (2011).</li><li>Alizadeh, A. M., Shiri, S., Farsinejad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+review:+from+bench+to+bedside.">Metastasis review: from bench to bedside.</a> Tumor. Bio.Volume. 35, 8483-8523 (2014).</li><li>Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+metastasis+predetermined.">Is metastasis predetermined.</a> Mol. Oncol. 1 (3), 263-264 (2007).</li><li>Ishiguro, T., Nakajima, M., Naito, M., Muto, T., Tsuruo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+genes+differentially+expressed+in+B16+murine+melanoma+sublines+with+different+metastatic+potentials.">Identification of genes differentially expressed in B16 murine melanoma sublines with different metastatic potentials.</a> Cancer Res. 56 (4), 875-879 (1996).</li><li>Clark, E. A., Todd, R. G., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC.">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406, 532-535 (2000).</li><li>Terranova, V. P., Hic, S., Diflorio, R. M., Lyall, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+metastasis.">Tumor cell metastasis.</a> Crit. Rev. Oncol. Hematol. 5 (2), 87-114 (1986).</li><li>Elkin, M., Vlodavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tail+vein+assay+of+cancer+metastasis.">Tail vein assay of cancer metastasis.</a> Curr Protoc in Cell Biol. , (2001).</li><li>Hagedorn, H. G., Bachmeier, B. E., Nerlich, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+degradation+of+basement+membranes+and+extracellular+matrix+and+their+regulation+by+TGF-beta+in+invasive+carcinomas+(Review).">Synthesis and degradation of basement membranes and extracellular matrix and their regulation by TGF-beta in invasive carcinomas (Review).</a> Int. J. Oncol. 18 (4), 669-681 (2001).</li><li>Hart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+selection+and+characterization+of+an+invasive+variant+of+the+B16+melanoma.">The selection and characterization of an invasive variant of the B16 melanoma.</a> Am J Pathol. 97 (3), 587-600 (1979).</li><li>Overwijk, W. W., Restifo, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N.P.+B16+as+a+Mouse+Model+for+Human+Melanoma.">N.P. B16 as a Mouse Model for Human Melanoma.</a> Curr Protoc Immunol. 20 (1), (2001).</li></ol>

저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

원거리의 모세관 침대에 자신을 확립 때때로 기본 성장 부위를 회피 전이를 나타내며, 혈액 또는 림프계 같은 경로를 통해 이주 꼬리 정맥 주사로 종양 세포 순환. 14이 프로토콜은 상술 한 차에 대한 모델로서 기능 전이. 주입 B16-BL6 셀의 최적의 수와, 실험자는 암세포 전이에 투여 약물 또는 면역 세포 집단, 후 중화의 상대적 효과를 결정할 수있다. 이 프로토콜은 한계가있다. 순환 성 종양 세포는 전이하는 능력으로 선택 및 주요 조직 적합성 복합체 클래스 1 분자의 낮은 레벨을 갖는, 따라서 비 - 면역 원성 하였다. (12) 또한, 전이의 다수의 갑작 클러스터 도입 환자에서 일반적인 시나리오 달리 여기서, 전이성 종양 세포의 수가 적은주 종양의 위치에서 장시간에 걸쳐 방출. 또한, 정맥 내로 도입 된 종양 세포는 조직 배양에서가 아니라 일차 종양 기원이다. 따라서 이러한 세포는 세포 접착, 마이그레이션, 면역 인식 및받는 기관 설립의 변화에 기인 보조 전이 달리합니다. (15, 16) 대안적인 프로토콜은 일차 종양 생성하고, 생성 된 &quot;자연&quot;폐 전이의 분석 B16 / BL6의 피하 주사 일 것이다. 이러한 &quot;자연&quot;전이가 자신의 &#39;실험&#39;에 비해 더 이질성을 포함하는 것으로 밝혀졌다, 더 밀접하게 인간 환자의 것과 일치. 프로토콜은 전이에 대한 실험적인 화합물의 영향을 판단 할 수있는 능력에 제한된다. 측면 꼬리 정맥 주입으로 혈류로 주입 된 흑색 종 세포의 개수는 H로 근사화 될 수있다emocytometer. &quot;자연스러운&quot;전이 그러나 폐 병소 생성 수에 추가 변수를 추가 종양 사이의 큰 얼룩이있다. 16 결론적으로 꼬리 정맥 B16-BL6 흑색 종 생쥐 모델은 전이의 치료 또는 면역 요법의 영향을 결정하기위한 간단한 모델을 나타낸다. 정맥 순환 성 종양 세포를 주입하여, 연구자들은 어느 정도, 환자의 전이 이후에 복제 할 수있다. 암 환자에서 전이성 종양 세포의 파괴적인 영향을 고려하면,이 모델은 전이의 다단계 과정을 이해하기위한 강력한 도구이다.

전이 악성 종양 환자에서 사망의 주요 원인이다. 암세포의 전이성 보급의 과정은 제대로 이해하지만, 전이, 적응의 사이트에있는 혈관에서 인접 조직의 침공, intravasation 림프계와 혈관, 생존에와 전위 순환 시스템 내에서, 혈관 외 유출 등 여러 단계를 포함 할 나타납니다 확산 및 보조 종양의 형성에 의해 새로운 사이트의 새로운 새 사이트의 미세하고 식민지에. 일이 생물학적 사건의 각각은 전이성 종양 세포의 변화, 보급 및 생존을 포함한다. 예를 들어, 세포 외 매트릭스와의 성공적인 상호 결합 간엽 표현형으로 종양 세포의 상피 세포의 표현형의 변화는, 인접한 조직에 침투하고, 신체의 다른 부위로 전이 할 수 있도록. 2,3 또한, 이들 전이를 세포체내 먼 부위에 이식 할 때 순환 혈류 내에서 생존하고 호스트로부터의 면역 감시를 회피한다. 4,5 마지막으로, 종양 세포는 이차 종양 증식 형성하기 위해 자신의 미세 환경에 적응한다. 6,7 따라서 전이는 암 환자에서 공통적 현상이지만, 전이성 종양 세포의 치료 개입 의무가있는 취약점 여러 영역을 갖는다. 흑색 종의 면역 원성 자연과 면역 최근 관심을 감안할 때, 흑색 종에 대한 모델. 점점 더 유용 혼자 미국에서 8, 흑색 종은 연간 약 9,000 명이 사망의 원인입니다. 흑색 종 전이의 9 일반적인 위치는, 뇌 뼈 있습니다 간, 폐. 먼 사이트로의 전이의 대부분은 혈류를 통해 발생한다. 혈액 암세포를 순환하는 면역 클리어런스를 회피 말단 기관의 모세 혈관 층에 도달해야성공적으로 자신을 확립하기 위해 혈관의 내피 세포를 통해 침입. 4-7,10 11 전이의 일반적이고 치명적인 현상을 모방하기 위해, 쥐 B16-BL6 세포주를 만들었습니다. 부모 C57BL / 6 흑색 종 세포 라인 B16-F0의 사망자는 B16-BL6은 방광 막 침투 6 연속 선택 다음 (B16-F10 결과) 정맥 주사에​​서 폐 전이 10 연속 선택의 최종 제품이다. 이와 같이 12는, 특히 정맥 내 주사 할 때 마우스의 전이성 병소의 확립에 대한 신뢰성 흑색 종 세포주이다. (13) 주입 및 B16 / BL6 세포의 성장을 허용하는 둘 이상의 주 뒤에 B57BL / 6 마​​우스의 꼬리 정맥에 B16-BL6 셀의 충분한 양을 주입 후 전이성 병소 폐에 형성된다. 해부 현미경에 의한 안락사 및 검사시의 수개별 초점 선물은 정량 할 수있다. 주입 B16-BL6 셀의 개수가 폐의 표면에 형성 병소의 수와 상관 관계로서 이는 차례로, 복용량 전이 효과를 확립하기 위해 사용될 수있다. 이 모델들은 알려진 전이성 세포 조사 폐, 간 또는 기타 기관에서 신속하고 예측 가능한 확산 설립을 용이 혈류에 직접 도입되는 &quot;실험 전이&quot;로 알려져있다. 이는 종양 세포는 종종 피하 주입 &quot;자발적 전이&quot;대조적이며, 유기 암세포 창고에서 전이가 발생한. 14,15 이는 생쥐의 꼬리 정맥으로 B16 / BL6 셀의 적절한 양을 주입하는 것이 중요하다. 너무 많은, 그리고 폐 전이로 덮여되고 주변 초점이 서로 구별 할 수 있습니다. 너무 적은 및 치료제의 영향으로 인해 본래 VARI으로 식별 할 수없는 것주사 사이의 관리 포인트. C57BL / 6 마​​우스의 폐에 병소의 최적의 수를 얻기 위해서는, 계정에 개인의 distinguishability하면서 폐의 표면에 설치 전이성 병소의 금액 주입 세포 수를 상관 할 필요 초점. 이 프로토콜은 C57BL / 6 마​​우스에 대한 전이 생쥐 흑색 종 모델을 시연 할 예정이다.

<table border="0" cellpadding="0" cellspacing="0" width="1227">
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			<td height="21" style="height:21px;width:377px;">Trypsin</td>
			<td style="width:275px;">Corning</td>
			<td style="width:288px;">MT25052CV</td>
			<td style="width:287px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium</td>
			<td>Corning</td>
			<td>MT15040CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase Contraast Hemacytometer</td>
			<td>Hausser</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Fine IV Insulin Syringes</td>
			<td>BD</td>
			<td>14-829-1D</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>22-363-750</td>
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			<td height="21" style="height:21px;">Mouse Restrainer</td>
			<td>Braintree Scientific</td>
			<td>NC9999969</td>
			<td>Restrainer choice depends on age/size of mice</td>
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			<td height="21" style="height:21px;">Heating Pad</td>
			<td>Harry Schein</td>
			<td>NC0012697</td>
			<td>Optional</td>
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</table>

injection of syngeneic murine melanoma cells to determine their metastatic potential in the lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

시각적 검사시, 초점면에서의 변화는 명백하다. 해부 현미경 종양의 수를 카운트하는 주입 종양 세포의 수와 생성 수 및 가산 초점 사이의 선형 관계를 생성한다. 주사 셀들의 최적의 개수를 사용하여, 목표는 62,500 개의 세포 / ㎖ 및 125,000 세포만큼 그들 /도 1a에 500,000 세포 / ml로하고, 너무 희박한 덮이지 같이 폐 완전히 포함되지 않는 하나 인 ml의. 이것은 또한 선형 상관 관계 (그림 1B)에 의해 정량화 할 수있다. <img alt="그림 1" src="/files/ftp_upload/54039/54039fig1.jpg" /> 꼬리 정맥으로 인한 그림 1. 폐 초점은 B16 / BL6 셀을 주조. (A) 폐 초점은 (화살표 아래 담당자 초점) 갈색 원형 대중으로 C57BL / 6 마우스에서 폐의 표면에 가시화 될 수있다. 굴로(각 이미지 위) 주입 된 세포 배양의 성만 때문에 (각 이미지 아래) 폐 초점을 결과의 해당 번호를 수행, 증가한다. 주입 된 세포의 가장 높은 밀도와 마우스에서 폐는 폐 병소의 가장 큰 시각 범위를 가지고있다. 스케일 바는 20mm를 나타낸다. 세포 배양 밀도 폐 병소의 상대 (B)을 열거. hemacytometer의 결정에 따라 125,000 세포 / ml 이상 거의 선형 관계가있다. <a href="https://www.jove.com/files/ftp_upload/54039/54039fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs at JoVE.com

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

동계 쥐 흑색 종 세포의 주입은 폐에서 그들의 전이성 잠재력을 확인하는 방법

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

injection-syngeneic-murine-melanoma-cells-to-determine-their

Research

JoVE Journal

Medicine

17.5K 조회수.  Beth Israel Deaconess Medical Center & Harvard Medical School. Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

비디오: Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

체외에서</em전이성 종양 성장에 휴면하고 스위치를 연구하기> 시스템

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

연구

의학

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

약시의 억제의 측정 및 치료

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

경쟁 손쥐 골수 이식 모델에서 기증자 세포 Engraftment를 확인하려면 수량 실시간 PCR을 사용하여

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

돼지의 심근 경색 및 기능적 결과의 평가

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

수정<em&gt; 시험관</em&gt; 침공 분석은 호르몬, 중재 암 세포 침략의 사이토 카인 및 / 또는 성장 인자의 잠재적 인 역할을 확인하는

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

형성 초점 : 세포 기반 분석은 유전자의 발암 성 가능성을 파악하기 위해

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

마우스 폐의 압력 볼륨 곡선의 측정

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

&quot;환자와 같은&quot;동소 이식 동계 마우스 모델 간세포 암의 전이

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

심장 수술 후 혈청 샘플의 영향을 건강한 자원 봉사자와 그들의 철새 잠재력에서 내피 전구 세포의 분리

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...